蛋清溶菌酶的改造及其抑菌活性

The Modification of Hen Egg-White Lysozyme and Its Antibacterial Activity

本研究旨在探讨高温酸性改造的蛋清溶菌酶对两种革兰阳性菌G^(+)(金黄色葡萄球菌Staphylococcus aureus、枯草芽胞杆菌Bacillus subtilis)和两种革兰阴性菌G^(-)(大肠杆菌Escherichia coli、鳗弧菌Vibrio anguillarum)的抑制作用。采用高温(57.0±0.1)℃、pH 2.0条件对蛋清溶菌酶进行改造,透射电镜观察改造蛋清溶菌酶的超微结构,8-苯胺基-1-萘磺酸(ANS)测定蛋清溶菌酶改造前后疏水性的变化,圆二色谱法与BeStSel软件分析改造后溶菌酶二级结构的改变,并采用牛津杯法测量改造后蛋清溶菌酶对供试菌的抑菌直径,生长曲线测定法测定改造后蛋清溶菌酶的最小抑菌浓度,Western blot检测4种供试菌菌液与菌体中溶菌酶含量变化。透射电镜结果显示,改造后蛋清溶菌酶为短杆状纤维结构;ANS和圆二色谱法结果显示,改造后蛋清溶菌酶疏水性增强,β折叠含量提高31.7%;牛津杯法显示,改造后蛋清溶菌酶对试验菌的抑菌强弱为鳗弧菌>枯草芽胞杆菌>金黄色葡萄球菌>大肠杆菌;生长曲线测定结果发现,鳗弧菌最小抑菌浓度为8μmol·L^(-1),大肠杆菌、金黄色葡萄球菌和枯草芽胞杆菌的最小抑菌浓度为6μmol·L^(-1);Western blot发现菌液中改造蛋清溶菌酶分子质量未发生改变,而菌体中溶菌酶含量和分子质量发生改变,革兰阳性菌和阴性菌均在10 ku处出现条带,且革兰阳性菌在22 ku处溶菌酶含量增加,但菌液上清均仅在14.3 ku处有条带。结果提示,改造蛋清溶菌酶对革兰阳性菌和阴性菌均具有较强的抑菌效果,为改造蛋清溶菌酶在畜牧业中的应用提供了基础数据。

The aim of this study is to modify the hen egg-white lysozyme(HEWL)and explore its effects on two gram-positive bacteria(Staphylococcus aureus,Bacillus subtilis)and two gram-negative bacteria(Escherichia coli,Vibrio anguillarum).The egg-white lysozyme was modified under the condition of(57.0±0.1)℃at pH 2.0,the ultrastructure of modified hen egg-white lysozyme was observed by transmission electron microscope,and the hydrophobicity of the egg-white lysozyme before and after the modification was measured by 8-anilino-1-naphthalenesulfonic acid(ANS).Circular dichroism and BeStSel software analysis were used to monitor the secondary structure changes of modified HEWL,Oxford cup method was used to measure the bacteriostatic diameter of modified egg-white lysozyme against experimental bacteria,and growth curve determination method was used to determine the minimum inhibitory concentration of modified egg-white lysozyme and Western blot was used to detect the changes of lysozyme content in the four experimental bacteria supernatant and cells.Transmission electron microscopy results showed that the modified egg-white lysozyme generated short rod-shaped fibril structures.ANS and circular dichroism detection results showed that the modified egg-white lysozyme was more hydrophobic and theβ-sheet content was increased by 31.7%.Oxford cup method showed that the inhibitory extent of the enzyme on the experimental bacteria was:Vibrio anguillarum>Bacillus subtilis>Staphylococcus aureus>Escherichia coli.The growth curve measurement results showed that the minimum inhibitory concentration was 8μmol·L^(-1) for Vibrio anguillarum,6μmol·L^(-1) for Escherichia coli,Staphylococcus aureus and Bacillus subtilis.Western blot found that the molecular mass of the modified egg white lysozyme in the bacterial solution was not changed,but the content and molecular mass of the lysozyme in the bacteria were changed.Gram-positive bacteria and negative bacteriaboth showed bands at 10 ku,and gram-positive bacteria showed increased lysozyme content at 22 ku,however the supernatant of the bacteria only had a band at 14.3 ku.These results indicated modified egg-white lysozyme had a strong antibacterial effect on both gram-positive bacteria and negative bacteria,which provided basic data for the application of modified hen egg-white lysozyme in the field of animal husbandry.